AOHUPO Membrane Proteomics Workshop
8 December 2006
Venue: University of Singapore, LT 32, Science Drive 4
This Workshop and the AOHUPO Membrane Proteomics Initiative are proudly
sponsored by GE Healthcare
9:00- 10:00 Session 1
9.00-9.20 Bill Jordan (Centre for Biodiscovery, Victoria University of Wellington,
Wellington) “The AOHUPO Membrane Proteomics Initiative”
9:20-9.40 Fuchu He (Beijing Proteome Research Center, Beijing Institute of Radiation
Medicine, and Institutes of Biomedical Sciences, Fudan University, Shanghai)
“Proteomic analysis of endoplasmic reticulum in C57BL/6J mouse liver”
9:40-10:00 Young-Ki Paik (Yonsei Proteome Research Center, Yonsei University,
Seoul) “Proteomic analysis of rat liver microsomal proteins by a liquid-based twodimensional chromatography combined with nano-LC-ESI-MS/MS”
10:00-10.30 Morning tea
10:30-11.30 Session 2
10:30-10:50 Paul Michael (Technology Manager, Molecular Biology GE Healthcare,
UK) “Multi-parameter cellular analysis”
10.50-11.10 Rudolf Grimm (Agilent Technologies Inc., Santa Clara, California) “A
novel strategy to enhance membrane protein identification by reversed-phase protein prefractionation and HPLC-Chip/MS”
11:10-11:30 Lifeng Peng (Centre for Biodiscovery, Victoria University of Wellington,
Wellington) “Proteomic analysis of a microsomal subcellular fraction from lactating
bovine mammary gland”
11.30-12:10 Session 3
11:30-11:50 Alice Len (Australian Proteomics Analysis Facility, Sydney) “Membrane
protein shotgun peptide IEF”
11.50-12:10 Robert Goode (Joint ProteomicS Laboratory, Ludwig Institute for Cancer
Research / Walter & Eliza Hall Institute of Medical Research and Department of
Biochemistry and Molecular Biology, University of Melbourne, Mellbourne) “The
cationic colloidal silica strategy for purification of adherent, integral, plasma membrane
proteomes from epithelial cells”
12:10 Discussion
“Membrane proteomics and the AOHUPO Membrane Proteomics Initiative”
1:00 End of Workshop
1:00 Lunch
ABSTRACTS
THE AOHUPO Membrane Proteomics Initiative
Bill Jordan, Lifeng Peng, Magalie Boucher, Graeme Lindsay, Pisana Rawson, Daniel
Klotz, Florian Dreyer
Centre for Biodiscovery and School of Biological Sciences, Victoria University of
Wellington, PO Box 600, Wellington 6140, New Zealand.
AOHUPO has chosen membrane proteomics as a research theme. This Membrane
Proteomics Initiative (MPI) is relevant to the larger goals of HUPO in characterising the
human proteome and to the biologically more diverse interests of the countries in the
AOHUPO region. There is therefore a human focus but also relevance to microbial, plant
and animal proteomics. This initiative was selected because of the importance and
challenge of membrane proteomics. Membrane proteins are calculated to represent a
large fraction of genomes, are essential in processes including signal reception and
transduction, are implicated in disease and are important targets for industry. Further
information is available at www.aohupo.org
Phase I of the MPI is technology development. A membrane standard has been prepared
for distribution to the participating laboratories. A range of proteomics methods will be
tested and compared with the goal of defining optimum analytical methods for the
various kinds of membrane proteins including trans-membrane proteins with multiple
membrane spanning domains. The MPI standard has been prepared from the large lobe of
livers from male 10-11 week old C57BL/6J mice. Preparation and distribution of this
standard has been generously supported by GE Healthcare.
Proteomic analysis of endoplasmic reticulum in C57BL/6J mouse liver
Yanping Song1, Ying Jiang1, Fuchu He 1,2
1. Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 33
Life Science Road, Beijing 102206, China.
2. Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China
Endoplasmic reticulum (ER) takes part in the protein synthesis, glycogenolysis and
biosynthesis of lipid and cholesterol. It also has a close relationship with membranebound and secretory proteins. For liver tissue, ER plays a great role in the
biotransformation of hydrophobic endogenous metabolites and xenobiotics. The disorder
of ER function can cause cell dysfunction and diseases. To gain a better understanding of
the critical role of ER function in liver, we present the ER proteome study on C57BL/6J
mouse liver. In contrast to other proteome studies we focused on the multiple
fractionation strategy to make full use of sample and to compare different subcellular
fractions from single homogenate as well. The purity evaluation method was also applied
to obtain highly purified organelle. Subsequently, ER proteins were separated by SDSPAGE prior to nano-LC-ESI-MS/MS analysis with gas phase fractionation by mass range
selection. 2018 and 1735 proteins were identified separately in rough and smooth
endoplasmic (rER, sER), in which 1232 and 1177 proteins matched by over two peptides.
Proteomic analysis of rat liver microsomal proteins by a liquid-based twodimensional chromatography combined with nano-LC-ESI-MS/MS
Hyoung-Joo Lee; Min-Seok Kown; Eun-Young Lee; Young-Ki Paik
Yonsei Proteome Research Center, Yonsei University, Seoul, Korea
A liquid-based two-dimensional separation system (2D-LC) method combined nano-LCESI-MS/MS was developed for analysis of hydrophobic rat liver microsomal proteins.
The membrane proteins were separated on chromatofocusing as neutral pH range (pH
8.5-4.0) and basic range(10.5-7.4) for the first dimension followed by non porous
reversed-phase HPLC for the second dimension. The detergent 2 % Trion X-100 was
used for efficient solubilization of membrane proteins and removal of soluble protein.
Nano-LC-ESI-MS/MS was used for large-scale identification of fractionated proteins.
Among 140 proteins identified by nano-LC-MS/MS, about 48% proteins were
hydrophobic proteins with more than one transmembrane domain (TMD) up to proteins
with 15 TMDs. Approximately 28 % of identified proteins was located on membrane
region. With respect to function, 28% proteins were identified to be involved in
metabolism, 14% protein were related to cell signal and regulation and 14% proteins
involved in transportation. The 2-D virtual image of pI vs. hydrophobicity could be used
for differential display analysis. Thus, our 2D-LC system can provide large-scale analysis
of hydrophobic rat liver microsomal proteins not only for the analytical separation but for
the preparative separation with high resolution as compared to traditional gel methods.
[supported by MOHW grant 03-PJ10-PG6-GP01-0002 (to YKP)]
Multi-parameter Cellular Analysis
Paul Michael
Technology Manager, Molecular Biology, GE Healthcare, UK
High-content analysis offers a range of approaches for high-throughput cellular analysis
providing an in-depth view of the effects of genes and drugs on biological processes. A
combination of automated imaging, sophisticated software and complementary
fluorescent biology are required to enable high-content analysis. This presentation will
cover validated biological assays and algorithms developed on robust imaging platforms.
A novel strategy to enhance membrane protein identification by reversed-phase
protein pre-fractionation and HPLC-Chip/MS
R. Grimm
Agilent Technologies Inc., 5301 Stevens Creek Blvd, Santa Clara, CA95052, USA
It is predicted that more than one third of the open reading frames in the human genome
are encoded with membrane proteins. Membrane proteins are involved in many crucial
biological processes and scores of examples have shown a direct link to human diseases,
thus there is an increasing interest to study membrane function and apply the knowledge
to drug discovery. However, analysis of membrane proteins represents a major challenge
in proteomics due to their amphipathic nature, and despite their biological importance
they remain an underrepresented subset of studied proteins within proteomics.
We have developed a strategy for enhancing membrane protein identifications by two
dimensional pre-fractionation and microfluidic nano-chip-LC/MS analysis. Specifically,
we have used a novel high-recovery macroporous reversed-phase (RP) C18 column to
pre-fractionate intact HeLa membrane proteins under high temperature conditions.
Collected RP fractions were further resolved by SDS-PAGE, in-gel digested and the
resulting tryptic peptides analyzed by HPLC-Chip/MS. Alternatively, RP fractions were
in-solution digested and tryptic digests analyzed via 2D HPLC-Chip/MS.
To date, we have identified more than 900 proteins with more than half of the
identifications predicted or verified as integral membrane proteins. This robust and highly
reproducible workflow has demonstrated utility for increasing membrane protein
identifications from a highly complex membrane protein sample. Optimized conditions
for the RP separation permitted total protein recoveries greater than 95%, enhanced peak
resolution and showed excellent reproducibility.
Proteomic analysis of a microsomal subcellular fraction from lactating bovine
mammary tissue
Lifeng Peng, Pisana Rawson, Danyl McLauchlan and T. William (Bill) Jordan
School of Biological Sciences, Victoria University of Wellington, Wellington, New
Zealand.
A proteomic investigation of a microsomal subcellular fraction from lactating bovine
mammary tissues was carried out using 2-DE with MALDI TOF MS and 1-DE with
RPLC ESI MS MS. 2-DE allowed 210 protein identifications, of which 45 (21.5%) were
microsomal proteins and 39 (18.6%) membrane proteins including 17 transmembrane
proteins. 1-DE revealed 723 proteins, of which 211 (29.3%) were microsomal proteins
and 204 (28.3%) were membrane proteins including 167 transmembrane proteins. From
the point of view of biological functions, 50 (23.2%) of the microsomal proteins are
involved in protein biosynthesis, 64 (30.4%) in protein folding, trafficking, assembly,
sorting and secretion and 22 (10.3%) were related to lipid metabolism, transport and
secretion, which is consistent with the prediction of the biological functions of
microsomes. Comparison of 2-DE and 1-DE results shows that the performance of 1-DE
in combination with RPLC ESI MS/MS was much greater than that of 2-DE with regard
to membrane proteins. The data reported here presented a comprehensive proteome
survey of the enriched microsomal fraction from the lactating bovine mammary tissues,
which provides knowledge for understanding molecular mechanisms in mammary glands.
Exploiting the membrane subproteome. A shotgun peptide immobilised pH gradient
– isoelectric focusing approach*
Joel Chick1, Mark Molloy1,2, Mark S. Baker1,2, Alice Len1,2
1
Dept. Chemistry & Biomolecular Sciences, Macquarie University, NSW, 2109,
Australia.
2
Australian Proteome Analysis Facility Ltd, Macquarie University, NSW, 2109,
Australia.
Membrane proteins play an important role in pathogenesis and are one of the most
exploited targets for therapeutic intervention. Proteomics is contributing to improving
the understanding of many disease processes at a molecular level. Despite this, traditional
proteomic methods used to analyse and identify membrane proteins remain a significant
bottleneck where there has been slow progress.
Overcoming physicochemical limitations dictated to by the hydrophobic nature of the
vast majority of membrane proteins is a key factor to enhancing the analysis of this
repertoire of proteins. We address this inherent limitation by employing a shotgun
proteomic approach. An established form of this technique involves tryptically digesting
a complex mixture of proteins and separating the generated peptides over single or
multidimensional liquid chromatography mass spectrometry.
Here, we present
preliminary data obtained from analysing rat liver membrane proteins utilising a peptide
immobilised pH gradient - isoelectric focusing coupled with this shotgun proteomic
approach.
Preliminary analysis of a total of 24 fractions of peptides focused over a pH range of 3-10
yielded an identification of 2957 non redundant peptides, corresponding to a total of 761
unique proteins with a false positive rate of 1%. 51 of these proteins have a GRAVY
score of > +0.3, validating the suitability of the approach for hydrophobic proteins that
are often found to be refractory to analysis by 2DE gels. Some examples of the rat liver
membrane proteins identified using this approach include putative transferrin receptor
protein 2 (TfR2), solute carrier family proteins and many cytochrome P450 genetic
variants, to name but a few. This study has illustrated that the majority of rat liver tryptic
peptides focused within the pH range of 3.3-5.2. Further in-depth analysis of the peptides
within this narrow IPG strip pH range will facilitate better peptide resolution and
subsequently enable further coverage of the membrane subproteome.
*
The authors would like to acknowledge GE Healthcare for their support as a industry
partner for the ARC linkage grant LP 0561182 that supports this project.
The cationic colloidal silica strategy for purification of adherent, integral, plasma
membrane proteomes from epithelial cells
Robert Goode1,2, Eugene Kapp1, Robert Moritz1 and Richard Simpson1,2
1 Joint ProteomicS Laboratory, Ludwig Institute for Cancer Research / Walter & Eliza
Hall Institute of Medical Research, Parkville, Victoria, Australia.
2 Department of Biochemistry and Molecular Biology, University of Melbourne,
Parkville, Victoria, Australia.
The plasma membrane is a desirable source for discovery of both biomarkers and drug
targets. However, purification of the plasma membrane specifically is often troublesome
due to the relative abundance of other cellular membranes and variability in membrane
density between cell lines. Additionally, classical membrane preparations are often
heavily contaminated with abundant basic proteins, such as histones and ribosomal
proteins (Simpson et al., 2000). Here we present a method for the purification of the
adherent plasma membrane from epithelial cell lines using cationic colloidal silica apical
membrane subtraction (Goode and Simpson, in press) and demonstrate its specificity for
integral plasma membrane proteins through detailed proteomic analysis. Of over 200
manually validated identifications, over 50% possess between 1 and 12 transmembrane
domains, including over 30 CD antigens and several markers of the basolateral plasma
membrane.
Goode, R.J.A. and Simpson, R.J. (in press). "Purification of basolateral integral
membrane proteins by cationic colloidal silica-based apical membrane
subtraction" in Proteomic analysis of membrane proteins: Methods and protocols
in Methods in Molecular Medicine series.
Simpson, R. J., et al. (2000). "Proteomic analysis of the human colon carcinoma cell line
(LIM 1215): Development of a membrane protein database." Electrophoresis
21(9): 1707-1732.